Compacted Hemostatic Cellulosic Aggregates

ABSTRACT

The present invention is directed to a hemostatic material comprising a compacted, hemostatic aggregates of cellulosic fibers. In some aspects, the hemostatic material further includes additives, such as carboxymethyl cellulose (CMC) or other polysaccharides, calcium salts, anti-infective agents, hemostasis promoting agents, gelatin, collagen, or combinations thereof. In another aspect, the present invention is directed to a method of making the hemostatic materials described above by compacting a cellulosic-based material into hemostatic aggregates. In another aspect, the present invention is directed to a method of treating a wound by applying hemostatic materials described above onto and/or into the wound of a patient.

FIELD OF THE INVENTION

The present invention is directed to flowable, bioresorbable hemostaticmaterials, particularly compacted aggregates of cellulose fibers, and tomethods for manufacturing such materials.

BACKGROUND OF THE INVENTION

In a wide variety of circumstances, animals, including humans, cansuffer from bleeding due to wounds or during surgical procedures. Insome circumstances, the bleeding is relatively minor, and normal bloodclotting functions in addition to the application of simple first aidare all that is required. In other circumstances substantial bleedingcan occur. These situations usually require specialized equipment andmaterials as well as personnel trained to administer appropriate aid.

Bleeding during surgical procedures may manifest in many forms. It canbe discrete or diffuse from a large surface area. It can be from largeor small vessels, arterial (high pressure) or venous (low pressure) ofhigh or low volume. It may be easily accessible or it may originate fromdifficult to access sites.

Conventional methods to achieve hemostasis include use of surgicaltechniques, sutures, ligatures or clips, and energy-based coagulation orcauterization. When these conventional measures are ineffective orimpractical, adjunctive hemostasis techniques and products are typicallyutilized.

The selection of appropriate methods or products for the control ofbleeding is dependent upon many factors, which include but are notlimited to bleeding severity, anatomical location of the source and theproximity of adjacent critical structures, whether the bleeding is froma discrete source or from a broader surface area, visibility and preciseidentification of the source and access to the source.

In an effort to address the above-described problems, materials havebeen developed for controlling excessive bleeding. Topical AbsorbableHemostats (TAHs) are widely used in surgical applications. TAHsencompass products based on oxidized cellulose (OC), oxidizedregenerated cellulose (ORC), gelatin, collagen, chitin, chitosan, etc.To improve the hemostatic performance, scaffolds based on the abovematerials can be combined with biologically-derived clotting factors,such as thrombin and fibrinogen.

Many products have been developed as adjuncts to hemostasis. Theseproducts include topical absorbable hemostats (TAH) such as oxidizedregenerated cellulose, gelatin in various forms with or without athrombin solution, and collagen powder, as well as biologically activetopical hemostatic products (topical thrombin solutions, fibrinsealants, etc.) and a variety of synthetic topical sealants.

One of the most commonly used topical hemostatic agents is SURGICEL®Original absorbable hemostat, made from oxidized regenerated cellulose(ORC). ORC was introduced in 1960 as a safe and effective hemostaticagent for many surgical procedures. ORC fabric has a loose knit in itsmatrix structure and conforms rapidly to its immediate surroundings andis easier to manage than other absorbable agents because it does notstick to surgical instruments and its size can be easily trimmed. Thisallows the surgeon to hold the cellulose firmly in place until allbleeding stops.

The control of bleeding is essential and critical in surgical proceduresto minimize blood loss, to reduce post-surgical complications, and toshorten the duration of the surgery in the operating room. Due to itsbiodegradability and its bactericidal and hemostatic properties,oxidized cellulose, as well as oxidized regenerated cellulose has longbeen used as a topical hemostatic wound dressing in a variety ofsurgical procedures, including neurosurgery, abdominal surgery,cardiovascular surgery, thoracic surgery, head and neck surgery, pelvicsurgery and skin and subcutaneous tissue procedures. A number of methodsfor forming various types of hemostats based on oxidized cellulosematerials are known, whether made in powder, woven, non-woven, knit, andother forms. Currently utilized hemostatic wound dressings includeknitted or non-woven fabrics comprising oxidized regenerated cellulose(ORC), which is oxidized cellulose with increased homogeneity of thecellulose fiber.

SURGICEL® absorbable hemostats are used adjunctively in surgicalprocedures to assist in the control of capillary, venous, and smallarterial hemorrhage when ligation or other conventional methods ofcontrol are impractical or ineffective. The SURGICEL® family ofabsorbable hemostats consists of four main product groups, with allhemostatic wound dressings commercially available from Ethicon, Inc.,Somerville, N.J., a Johnson & Johnson Company:

SURGICEL® Original hemostat is a white fabric with a pale yellow castand a faint, caramel like aroma, this material is strong and can besutured or cut without fraying;SURGICEL® NU-KNIT® absorbable hemostat is similar to Original but has adenser knit and thus a higher tensile strength, this material isparticularly recommended for use in trauma and transplant surgery as itcan be wrapped or sutured in place to control bleeding;SURGICEL® FIBRILLAR™ absorbable hemostat form of the product has alayered structure that allows the surgeon to peel off and grasp withforceps any amount of material needed to achieve hemostasis at aparticular bleeding site, may be more convenient than the knitted formfor hard to reach or irregularly shaped bleeding sites and isparticularly recommended for use in orthopedic/spine and neurologicalsurgery;SURGICEL® SNoW™ absorbable hemostat form of the product is a structurednon-woven fabric that may be more convenient than other forms forendoscopic use due to the structured, non-woven fabric and is highlyadaptable and recommended in both open and minimally invasiveprocedures.

Other examples of commercial resorbable hemostats containing oxidizedcellulose include GelitaCel® resorbable cellulose surgical dressing fromGelita Medical BV, Amsterdam, The Netherlands. The commerciallyavailable oxidized cellulose hemostats noted above are available inknitted, nonwoven fabrics or powder form. Additional hemostaticproducts, such as powders consisting of microporous polysaccharideparticles and plant starch based particles, are also commerciallyavailable as Arista and Perclot.

U.S. Pat. No. 8,815,832 discloses a hemostatic material comprising aball milled compacted ORC powder comprising particles having averageaspect ratio from about 1 to about 18, said powder having tapped densityof at least 0.45 g/cm³, an average size of 1.75 microns to 116 micronswith a median size of 36 microns and a flowability of at least 7.5 cm/s.

U.S. Pat. No. 3,364,200 to Ashton and Moser describes a resorbable,surgical hemostat in the form of pledgets of integrated oxidizedcellulose staple fibers.

U.S. Patent Publication 2008/0027365 to Huey describes an apparatus forpromoting hemostasis utilizing oxidized cellulose in the form of acompressible, shapeable mass that is formed into a sheet for placementon a bleed site and further having a sleeve in a form of a tubular shelldimensioned to receive a limb.

U.S. Patent Publication 2004/0005350 to Looney et al. discloseshemostatic wound dressings utilizing a fibrous fabric substrate madefrom carboxylic-oxidized cellulose and containing a porous, polymericmatrix homogeneously distributed through the fabric and made of abiocompatible, water-soluble or water-swellable cellulose polymer,wherein the fabric contains about 3 percent by weight or more ofwater-soluble oligosaccharides.

PCT Patent Publication WO 2007/076415 by Herzberg et al. and entitled“COMPOSITIONS AND METHODS FOR PREVENTING OR REDUCING POSTOPERATIVE ILEUSAND GASTRIC STASIS”, discloses milling of ORC, particularly cryogenicmilling, using a cutting blade of a motor-driven mill.

An article titled “The Ball-Milling of Cellulose Fibers andRecrystallization Effects”, Journal of Applied Polymer Science, Volume 1Issue 3, Pages 313-322, (1959) by Howsmon and Marchessault, reportsresults of a study of the effect of fine structure on thedecrystallization process which results from the ball-milling ofcellulose. The rate of decrystallization is sensitive to the type offine structure and is accelerated by the presence of moisture. Theextent of chain degradation was greater in air atmosphere than in carbondioxide, suggesting that mechanically induced free radical degradationoccurs along with other chain-breaking processes. A study of the densityand moisture regain of the samples after various times of milling showedthat a linear relation between regain and density held over the entirerange studied. The relation was the same for native and regeneratedcellulose. The process of recrystallization of the ball-milled sampleswas studied under various conditions and compared to the hydrolyticallyinduced recrystallization of rayons. The reference discloses effect offine structure on the decrystallization process which results from theball-milling of cellulose fibers.

U.S. Pat. No. 6,627,749 discloses a process for grinding oxidizedcellulose using a pestle and mortar or in a ball mill or any otherconventional laboratory grinder. It further discloses that when cottonlinter sheet is used as the starting cellulose source, the fiber lengthof the product decreases with increasing reaction time. Whenball-milled, the long fibrous structures of the product turn intosmaller fibers, to loosely-packed spherical aggregates. No significantchange in the crystallinity of these samples occurs as a result of ballmilling. The reference discloses long fibrous oxidized cellulose ballmilled to form small fibers or loosely packed spherical aggregates.

Other related references include: U.S. Pat. No. 6,309,454, “Freeze-driedcomposite materials and processes for the production thereof”; U.S. Pat.Nos. 5,696,191; 6,627,749; 6,225,461 to Kyoko et al.; PCT PatentPublication WO2001/024841 A1, Compositions for the Treatment of WoundContracture; and European patent publication EP1,323,436 to Dae Sik etal.

Other related references include: An article titled “The role ofoxidized regenerated cellulose/collagen in chronic wound repair and itspotential mechanism of action”, The International Journal ofBiochemistry & Cell Biology 34 (2002) 1544-1556, Breda Cullen et al.; anarticle by Rangam et al. teaching methods of making silk powders throughmilling processes [Powder Technology 185 (2008), p 87-95]; an article byYasnitskii et al., Oxycelodex, a new hemostatic preparation,Pharmaceutical Chemistry Journal, 18, 506-508; discloses an Oxycelodexpaste that consists of two components, oxidized cellulose powder and a20% aqueous solution of dextran.

U.S. Patent Publication 2006/0233869 to Looney et al. discloses using achopping or shredding process to make ORC micro-fibers from ORC fabrics.The rod-like shaped fibers had sizes which ranged from about 35 to 4350micrometers.

There is a need in improved hemostatic forms and materials whichfacilitate ease of application and rapid onset of hemostasis.

SUMMARY OF THE INVENTION

The present invention is directed to a hemostatic material comprising acompacted, hemostatic aggregates of cellulosic fibers. In some aspects,the hemostatic material further includes additives, such ascarboxymethyl cellulose (CMC) or other polysaccharides, calcium salts,anti-infective agents, hemostasis promoting agents, gelatin, collagen,or combinations thereof. In another aspect, the present invention isdirected to a method of making the hemostatic materials described aboveby compacting a cellulosic-based material into hemostatic aggregates. Inanother aspect, the present invention is directed to a method oftreating a wound by applying hemostatic materials described above ontoand/or into the wound of a patient.

The present invention is also directed to method of making a pluralityof hemostatic aggregates milling a cellulosic source material to form anintermediate fine fibers; humidifying the intermediate fine fibers;roller compacting the intermediate fine fibers to form hemostaticaggregates; sieving the hemostatic aggregates; dehumidifying thehemostatic aggregates; and optionally dosing the resulting hemostaticaggregates into storage containers or into delivery devices. The millingstep can be preceded by a step of slitting and cutting the cellulosicsource material forming pieces. The milling step can be a two-partprocess with the second part is performed in an air classifier whereinthe second part can be repeated three times. The intermediate fine fiberpreferably has a size distribution with d50 of less than about 100microns and d90 of less than about 180 microns. The intermediate finefibers can be humidified to water content of between 11.0% and 20% byweight. The intermediate fine fibers can be roller compacted materialand then subjected to pre-breaking and subsequently followed by a stepof final milling. The intermediate fine fibers are preferably compactedat a roller pressure of at least 130 bars. The intermediate fine fibersare preferably compacted at a roller force of at least 26.0 kN/cm. Theresulting materials are selected to produce a targeted hemostaticaggregates fraction having dimensions along their longest axis of 75-300μm by screen sieving method. Preferably, the targeted hemostaticaggregates fraction is characterized by a size distribution such thatd15 is greater than about 80 microns, d50 is from about 140 to 250microns and d90 is less than about 370 microns. The hemostaticaggregates intended for dosing preferably having a moisture content ofloss on drying of less than about 5%, more preferably less than 2%. Thesource materials can be selected from oxidized regenerated cellulosicfabric, oxidized regenerated cellulose non-woven fabric, shreddedoxidized regenerated cellulosic material or combinations thereof. Thesource materials can further comprises an additive selected from thegroup consisting of carboxymethyl cellulose, calcium salt, ananti-infective agent, a hemostasis promoting agent, gelatin, collagen,or combinations thereof. The present invention further relates to amethod of treating a wound by applying the hemostatic aggregatesprepared as described above onto and/or into the wound of a patient.

The present invention further relates to hemostatic particulateaggregates composed of a plurality of interconnected individualcellulosic fibrils that in aggregate form have a sphericity of at least0.5, a diameter along its longest axis that is less than about 500microns and greater than about 50 microns. The hemostatic aggregates canalternatively be expressed as having a size distribution profile withd15 greater than about 80 microns, d50 from about 140 to 250 microns,d90 less than about 370 microns, a bulk density greater than 0.45 g/mL,and sphericity (sh50) equal or greater than 0.70. The hemostaticaggregate preferably are characterized by having substantially no sizedistribution changes or minimal size distribution changes when subjectedto a vibratory challenge, more preferably the size distribution profileof the hemostatic aggregates as measured by d50 does not fall below 100microns. In one embodiment, the size distribution changes arecharacterized by a QICPIC optical sensor at 0.2 bars. In a still furtherembodiment, the size distribution changes or minimal size distributionchanges are based to processing at 1.0 bar vacuum.

The present invention further relates to hemostatic aggregates that havebeen milled, humidified, roller compacted, and dried cellulosicmaterial. The present invention further relates to methods of treating awound by applying the hemostatic aggregates as described above ontoand/or into the wound of a patient.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic diagram of the manufacturing process.

FIG. 2 is a graph showing a series of size distribution curves.

FIG. 3 is a graph showing a series of size distribution curves.

FIG. 4 is a graph showing a series of size distribution curves.

FIG. 5 is a graph showing performance of selected materials.

FIG. 6 is a graph showing performance of selected materials

FIG. 7 is a graph showing performance of selected materials

DETAILED DESCRIPTION

The inventors discovered a process for making hemostatic aggregateshaving surprising properties and highly beneficial effects forhemostasis. Hemostatic aggregates according to the present invention aremade from oxidized cellulose-based fiber materials or from pre-shreddedoxidized cellulose-based materials, whereby the resulting hemostaticaggregates can be used for various surgical and wound healing topicalapplications, such as anti-adhesion barriers, hemostats, tissuesealants, etc. Oxidized regenerated cellulose materials that can be usedas a starting material for making the hemostatic aggregates of thepresent invention are known and commercially available. The startingmaterials can include absorbable woven or knitted fabric or non-wovenmaterials comprising oxidized polysaccharides, in particular oxidizedcellulose and the neutralized derivatives thereof. For example, thecellulose may be carboxylic-oxidized or aldehyde-oxidized cellulose.More preferably, oxidized regenerated polysaccharides including, butwithout limitation, oxidized regenerated cellulose may be used. Oxidizedregenerated cellulose is preferred due to its higher degree ofuniformity versus cellulose that has not been regenerated. Regeneratedcellulose and a detailed description of how to make oxidized regeneratedcellulose are set forth in U.S. Pat. Nos. 3,364,200, 5,180,398 and4,626,253, the contents of each of which are hereby incorporated byreference as if set forth in its entirety.

Examples of preferred cellulosic materials that may be utilized include,but are not limited to, INTERCEED® absorbable adhesion barrier,SURGICEL® Original absorbable hemostat, SURGICEL® NU-KNIT® absorbablehemostat, SURGICEL® FIBRILLAR™ absorbable hemostat, SURGICEL® SNoW™absorbable hemostat.

Hemostatic aggregates of the present invention can perform as a hemostatin either a paste or powder form with superior hemostatic properties andgood tissue conformability and flowability. In addition, hemostaticaggregates can be physically incorporated with other agents andbiopolymers to improve adherence to tissues, sealing properties, and/oranti-adhesions properties.

In one aspect of the present invention, there is provided a method formaking hemostatic aggregates having beneficial hemostatic, woundhealing, and other therapeutic properties. A preferred method of thepresent invention is applied to manufacture hemostatic aggregatesdirectly from cellulosic materials, such as ORC fabric or non-wovenproducts such as these discussed above.

Briefly, a preferred manufacturing process starts with ORC material,such as SURGICEL® Original absorbable hemostat, as which is cut into 1-to 2-inch wide sections before the material is fed into a blade thatcuts the fabric into smaller pieces. The cut ORC fabric pieces are thenground into intermediate ORC fine fibers by two consecutive millingprocesses (hammer milling and air classifier milling). In an alternativeembodiment, the cut ORC fabric pieces are converted directly intointermediate fine fibers in a ball mill. The resulting intermediate ORCfine fibers are then humidified to about 11% to about 16% as measured byOhaus halogen moisture analyzer and then roller compacted into largeraggregates. The moisture analyzer operates on a thermogravimetricprinciple wherein the moisture analyzer determines the weight of thesample; the sample is then quickly heated by the integral halogen dryerunit and moisture vaporizes. During the drying operation, the instrumentcontinuously determines the weight of the sample and displays theresult. On completion of drying, a tabulated result is displayed aspercent moisture content, percent solids, weight or percent regain, inparticular, the analyzer tests between 0.5-1 grams of aggregate with afour (4) minute ramp, 90C maximum temperature and the followingsettings: Test ID—LOD; Profile—Standard; Dry Temperature—90C; SwitchOff—A60; Result—Moisture %; Custom—Off; Target Weight—None. Sieving ispreferably done to separate target particles between the size of 75 and300 microns determined by screen sieving.

Excess moisture introduced for purposes of compaction is removed by adehumidification or drying process after compaction and sieving step forsubsequent dosing into applicator devices and then subjected to thedevice packaging and sterilization. Preferred storage moisture prior todosing into an applicator is preferably less than about 2% at conclusionof drying to achieve preferably less than 6% moisture content incontrolled environment (0.3-0.6%/hr per 500 gram sample moisture gaindepending on relative humidity, commonly 25-55% relative humidity) fordosing into applicators.

More specifically, one process for manufacturing the inventivehemostatic aggregates comprises the steps of: a) slitting and cutting ofcellulosic source material; b) milling the resulting material from stepa); c) a second milling step in an air classifier; d) humidification; e)roller compaction; f) sieving; g) dehumidification or drying; h)optional dosing into storage containers or into delivery devices,primary packaging and secondary packaging; and i) optionalsterilization.

Slitting and cutting can preferably be performed to slit and cut fabricinto appropriate size pieces that are between approximately 1 inch by 3inches or 2 inches by 3 inches, though smaller pieces can also be used.The main operations performed for slitting and cutting are to unwind aroll of fabric, slit the fabric into strips, cut the strips to size anddeliver the cut pieces into the first milling step. A number of cuttingand slitting machines are known and commercially available, such as AZCOModel FTW-1000 available from AZCO.

In the first milling step, processed pieces of cellulosic fabric areconverted from an intermediate coarse fiber produced in the slitting andcutting step to a material having a D90 value of less than 452 μm andD50 value of less than 218 μm, while having minimal impact on the colorindex and water soluble content of the material. A number of machinesfor milling are commercially available, such as Models DASO6 andWJ-RS-D6A manufactured by Fitzpatrick, which are hammer mill typemilling machines, equipped with a 497 micron round screen and a set ofblades that breaks down the fabric until it passes through the screen toproduce intermediate coarse cellulosic fiber. In an exemplary processingrun, mill speed can be about 7000 RPM; processing temperature at lessthan 80° C.; screen size between 1534 and 9004; number of blades as 8 (2impellers each); blade type as a 225 knife, impact type blades; bladeorientation set as “impact”.

Size distribution D50 is also known as the median diameter or the mediumvalue of the aggregate size distribution, it is the value of theaggregate's diameter at 50% in the cumulative distribution. For example,if D50 is 218 μm, then 50% of the aggregates in the sample are largerthan 218 μm, and 50% are smaller than 218 μm. Size distribution is thenumber of aggregates that fall into each of the various size rangesgiven as a percentage of the total number of all sizes in the sample ofinterest. Accordingly, D90 value refers to 90% of aggregates having asize that is smaller than the D90 value, while D10 refers to 10% ofaggregates having a size smaller than the D10 value.

At this stage in the preferred process, the size of the intermediatecoarse fiber produced in the first milling step is further reduced to aD90 value of less than 177 μm and a D50 value of less than 95 μm whilekeeping a minimal impact on the color index and water soluble content ofthe material. A number of machines are available for second millingstep, such as an Air Classifier/F10 Quadro Fine Grind from Quadro.

Intermediate coarse fiber from the first milling step can be fed at acontrolled rate into the second mill and passed through two millingchambers that are separated by a milling screen. The material can bepulled through the milling chamber by an air blower. The intermediatecoarse fiber can be processed through the air classifier equipment threetimes in order to obtain the desired size. At the end of the secondmilling step, the intermediate fine fiber can be collected.

In an exemplary processing run, a Quadro Air Classifier F10 can be usedin the second milling step with a milling speed of 8400 rpm, blowerspeed of 1800 rpm, 0.0018″ round hole screen and 3 passes. ORCintermediate fine fiber can be also produced in one step by ball millinginstead of the two steps milling steps as described above. In analternative ball milling embodiment, 50 g of pre-cut ORC fabric (2″×2″)is ball milled with 12 high-density Zirconia (zirconium dioxide ZrO2, 20mm in diameter; Glen Mills Inc., Clifton, N.J., USA) by placing theballs and the samples in a 500 mL grinding jar. The jar is clamped intothe latching brackets and then counterbalanced on the planetary ballmill PM100; Retsch, Inc., Newtown, Pa., USA). The milling is thenperformed bi-directionally at 450 rpm for 20 minutes.

Following the milling process, the resulting cellulosic intermediatefine fiber is humidified to a moisture content between preferably about11% and about 18%, more preferably between 11% and about 16%, mostpreferably about 12-16% for the subsequent processing, including aroller compaction process. A preferred humidity chamber suitable for thehumidification step is commercially available as ModelCEO-916-4-B-WF4-QS by Thermal Product Solutions. Humidification ofchamber air is achieved by water vapor injection. The typicalsteady-state temperature of 25° C. can be utilized, while the humiditylevel can be cycled between 75% and 85%, with a preferred target of 85%air humidity. Humidification time or residence time of the materialinside the humidity chamber can range from several hours to several daysdepending on the quantity of the material and air recirculation. In atypical and preferred cycle, the material will have 12-13 hoursresidence time for about 3,000 grams of cellulosic intermediate finefiber arranged in several trays and exposed to 85% relative humidity anda target of 12% moisture content of the powder after humidification.

Use of cellulosic intermediate fine fiber with a moisture content fedinto the compaction step that is greater than 16%, such as a moisturecontent of 20% by weight, the resulting ORC intermediate fine fibercaked during compaction, exhibited very poor flowability, and jammed thecompactor. Thus, high humidity of the intermediate fine fiber does notresult in suitable hemostatic aggregate materials. Conversely, when themoisture content of the intermediate fine cellulosic fiber is lower thanabout 8%, the yield of hemostatic aggregates is extremely low, somewhereabout 5% yield of desired hemostatic aggregates.

Humidified intermediate fine ORC fiber is then compacted and sieved toobtain hemostatic aggregate materials. The roller compactor compacts thefeed, which is then subjected to pre-breaking, final milling and sievingin a screener to obtain the desired hemostatic aggregates sizes.

Compaction equipment is known and commercially available. Exemplarycompaction units are the Fitzpatrick Chilsonator IRR220-L1A with Retschmanual sieving AS200 Screener and the Fitzpatrick Chilsonator CCS220/M3B& RV-M5A with Screener Sweco Vibro-energy unit integrated under M5A. Thecompaction processing can be performed using two separate subsystemsthat are bound by a common electrical system. For example, a firstsubsystem (Roller Compactor: main unit) can be the FitzpatrickChilsonator CCS220 roller compactor and the M3B mill for pre-breakingthe compacted material, while the second subsystem (Roller Compactor:secondary milling unit) is M5A mill for the final milling with a Swecoor Retch screener for the separation to obtain the desired sizeaggregates.

Humidified intermediate fine cellulosic fiber can be fed into the hopperof the roller compactor unit, first passed through a main milling unitand then proceed on through a second milling unit. A container can beprovided that captures the pre-broken cellulosic material resulting fromthe main milling unit. The pre-broken pieces of cellulosic material canthen be fed into the secondary milling unit, which performs the finalmilling and screening utilizing a screen mesh. The resulting milledcellulosic material is preferably separated into fines (<75 μm), targets(75-300 μm), and overs (>300 μm) using a screen mesh, such as the Swecoor Retch screener described above.

Referring to Table 3, testing showed that by using a lower size, asmeasured by d(50) and/or d(90), for the intermediate fine cellulosicfibers from the second milling step, resulted in aggregate product fromthe compactor sequence has spherical value that approaches 1. A higherfiber moisture content (16% LOD intermediate fine fibers as measured byOhaus MB45 moisture analyzer resulted in resulting aggregates having ameasured sphericity of 0.76. In contrast, when the moisture content forthe intermediate fine fibers was around 11% LOD, the resultingaggregates had a sphericity of 0.72. Higher moisture content ofintermediate ORC fibers results in higher sphericity of ORC compactedaggregates.

Preferred process parameters for the roller compaction and sievingprocesses are as follows: Roller Pressure about 125-135 bar, with targetof 130 bar; Roller Speed about 3 RPM; Roller—diamond knurl; Startingmaterial sizes are d50 less than about 95 microns and d90 less than 177microns; Starting Moisture Content is greater than about 11% but lessabout 16%; Roll Force Values about 26.0 kN/cm; Horizontal feed screwspeed about 19 rpm, vertical feed screw speed about 265 rpm; Sievingseparated target hemostatic aggregates (d90 less than 370 microns, d50between 140-242 microns and d15 higher than 86 microns. The preferredroller pressure is higher than levels typically used on rollercompactors and produced materials having aggregate durability asdemonstrated following vibratory challenge.

Cellulosic intermediate fine fiber batches were tested with differentroller compaction systems. Of the tested systems, only the FitzpatrickCCS20/M3B and IRR220-L1A models produced acceptable hemostaticaggregates. Without being held to any particular theory, it is believedthat these preferred units were able to operate at sufficient rollerforce (26 kN/cm) and with a vertical orientation of the feed to thecompaction rolls.

Moisture is removed from hemostatic aggregates that are obtainedfollowing roller compaction and sieving in a dehumidification or dryingstep. The dehumidification or drying step preferably does notsignificantly affect any other product quality attributes, such ascolor, bulk density, water soluble content, size, and sphericity.Typically, 750 grams or less of the powder can be dried as a batch usinga conventional fluidized air bed. The resulting dried powder can bepacked and stored in sealed foil pouches. Dehumidification equipment isknown and commercially available. An exemplary bench-top fluidized airbed is commercially available from Retsch (TG-200) with 6L capacity.Alternatively, a fluidized bed Model No. 0002 from Fluid Air (Aurora,Ill.) can also be used.

Example 1. Manufacturing and Characterization

Hemostatic aggregates were made from ORC material as described abovethrough steps of slitting and cutting of ORC source material usingSURGICEL® Original fabric including a first milling step, a secondmilling step via air classifier to obtain an intermediate fine ORCfiber, humidification of the intermediate fine ORC fiber, rollercompaction, granulating, sieving and dehumidification.

Hemostatic aggregate materials comprise a plurality of individualfibrils of fine ORC fiber that have been compacted and joined togetherby a compaction process. In preferred aspects, the hemostatic aggregatematerials comprise at least 5 elongated individual fibrils of fine ORCfiber, more preferably at least 10 elongated individual fibrils of fineORC fiber, or between 5 and 100 elongated individual fibrils of fine ORCfiber, such as 10-50.

The resulting materials are aggregates, not particles. There is no coreregion or defined pores. Rather, the fibrils or fibers appear to form aninterlocking web without loss of their fibril structure, eachinterconnected at discreet points. The processes described above produceaggregate having a fibril-interconnected structure with sufficient bulkto have connections and fibers to provide greater density than plasma,and strength to sink and then readily disperse to maximize thecoagulating effects of the carboxylic groups.

Hemostatic aggregates of the present invention have an overall size (asdetermined by their largest dimension) of less than about 500 microns,but generally larger than about 50 microns. Hemostatic aggregatematerials with such dimensions should comprise the majority of theparticles constituting the final hemostatic material, i.e. over 50%,such as over 80% or over 90% of all particles. Preferred inventivehemostatic aggregate materials are characterized by a size distributionsuch that [d15>86 microns], [d50, 140˜242 microns], [d90<370 microns] asmeasured by QICPIC FERET_MIN Q3 method. QICPIC is a high speed imageanalysis sensor available from Sympatec GMBH, Germany.

Bulk density is the ratio of the mass of an untapped powder sample andits volume including the contribution of the interparticulate voidvolume. Bulk density measurement was performed by following USP 616(2012). Inventive hemostatic aggregate materials preferably have a bulkdensity (g/mL) within the range 0.3 to 0.7, preferably greater than 0.45g/mL, such as 0.5 g/ml.

Sphericity (sh50) of the median particles (D50) was equal or greaterthan 0.5 by Sympatec QICPIC method, such as 0.70, where 1 corresponds toa sphere, indicating that the hemostatic aggregates have a relativelyspherical shape. Sphericity was defined and measured as shown below.Sphericity of hemostatic aggregates is related to the diameter of acircle that has the same area as the projection area of the aggregate.The sphericity, S, is the ratio of the perimeter P of the equivalentcircle, P_(EQPC), to the real perimeter, P_(real). For A=area of theparticle, the sphericity is defined by the formula below:

$S = {\frac{P_{EQPC}}{P_{real}} = \frac{2\sqrt{\pi \cdot A}}{P_{real}}}$

The resulting sphericity has a value between 0 and 1. The smaller thevalue, the more irregular is the shape of the particle. This resultsfrom the fact that an irregular shape causes an increase of theperimeter. The ratio is always based on the perimeter of the equivalentcircle because this is the smallest possible perimeter with a givenprojection area. The value of 1 corresponds to a perfect sphere.

Several sizes of hemostatic aggregate materials were developed andtested and compared to fine intermediate ORC fiber with differingparticle size as shown in Table 1 below.

TABLE 1 Comparison of ORC based hemostatic powders and hemostaticaggregates tested. Size Range (sieve method, Bulk Name Productionmicrons) density Observations ORC Fine Ball Milled ORC Fine 0.57 g/mLToo dusty, Fiber or Hammer Fiber 1 0.43 g/mL floats on milled, (36.5 μm,d50 by blood and shredding, DLS (Dynamic has poor no Light Scattering)efficacy compaction ORC Fine Fiber 2 (62 μm, d50 by DLS) HemostaticHammer 600 μm~800 μm N/A Good Aggregates Milled, then hemostasis Largeroller efficacy, compacted however granules are too large to spray,difficult to deploy onto the wound by spraying Hemostatic Hammer 106μm~425 μm 0.41 g/mL Good Aggregates milled hemostasis Coarse or ballefficacy and milled, then ability to roller adhere to the compactedwound site Hemostatic Hammer 106 μm~300 μm 0.37 g/mL Good Aggregatesmilled hemostasis Fine or ball efficacy and milled, then ability toroller adhere to the compacted wound site

If the compaction force is too low e.g. below about 10 kN/cm, theresulting material will return to its original state as a fine fiber inthe granulator associated with the compaction system (post-compaction orsecondary milling). If the compaction force is too high, the productwill be “overpressed”. Overpressing was observed when the material comesout of the roller compaction process, such as discolored extremely hot,or severely cracked. When using process parameters as defined above andusing a vertical screw speeds of higher than 22 rpm, the compactedribbon showed signs of burning, thereby thermally damaging thecellulosic material.

As described above, hemostatic aggregates are created by forcing ORCfine powder particles under pressure between two-counter rotating rollsto produce ribbon-like “compacts” that are then milled into aggregates,which are subjected to sieving to obtain desired hemostatic aggregatesbetween 106 μm and 300 μm by screen sieving.

Again, without intending to be bound to any particular theory, thebonding mechanisms that may hold the particles together are (1) van derWaals' forces—during compaction, the ORC material is squeezed so thatthese van der Waals' forces bind all the material together to form asolid compacted aggregates and (2) and inter-molecular hydrogen bondingto bring all the material together as well when certain level ofmoisture is present.

Example 2

Using the manufacturing techniques explained above, hemostatic aggregatesamples were prepared with and without humidification step; all otherprocessing steps being equal. Both specimens were exposed to sizedistribution measurement by using Sympatec QICPIC equipment using 0.2bar vacuum processing, and the size distribution curves were obtained(FIG. 1). Curve 1 shows size distribution of the specimen made with ahumidification step applied to ORC intermediate fine fiber prior toroller compaction, while curve 2 shows size distribution of the specimenthat was made without the humidification applied to ORC intermediatefine fiber.

After that both specimens 1 and 2 were exposed to a vibratory test. Thetest consisted of positioning vials containing 2 g of hemostaticaggregates powder on a sieve shaker (Retch AS200) which vibrated atamplitude of 1 mm/g for 90 minutes, followed by 3 mm/g for 90 minutes.After the vibratory challenge the specimens were again exposed to thesame size distribution measurement by Sympatec QICPIC. The results arealso shown in FIG. 2.

Curve 1 a of FIG. 2 shows size distribution of the specimen made with ahumidification step and exposed to a vibratory test. Curve 2 a of FIG. 2shows size distribution of the specimen made with no humidification stepand exposed to the same vibratory test. It can be seen that the controlspecimen 2 which was made of ORC intermediate fine fiber not subject tohumidification prior to roller compaction exhibited a significant changein size distribution from which the size decreased indicating breakageof the hemostatic aggregates into smaller subunits, with d50 changingfrom 137 microns to 50 microns. On the contrary, specimen 1 shows noappreciable change as curves 1 and 1 a are very similar. The moisturecontent of humidified ORC intermediate fine fiber used to makehemostatic aggregates specimens having size distributions shown incurves 1 and 1 a was within 11-16%. The moisture content of ORCintermediate fine fiber used to make hemostatic aggregates specimensshown in curves 2 and 2 a was 2.0%. The significant change in propertiesas a result of the vibratory challenge is undesirable and may result inadverse effect on therapeutic efficacy, as will be shown below. Thevibratory challenge indicates dosing, storage and transportationchallenges that hemostatic aggregates can be subjected to in use, andthus a significant change in properties can result, with detrimentaleffect upon hemostatic efficacy. Advantageously, according to one aspectof the present invention, hemostatic aggregates have substantially nosize distribution changes or minimal size distribution changes aftersubjected to a vibratory challenge, as measured by Sympatec QICPICoptical sensor at 0.2 bars.

Example 3

A test was performed using the methodology described above for sizedistribution measurements. As above, hemostatic aggregates samplesprepared with and without humidification step, with all other processingsteps being equal, were measured on the same QICPIC equipment but usingtwo pressure settings—low pressure of 0.2 bar vacuum, and elevatedpressure of 1 bar vacuum. Each specimen was exposed to size distributionmeasurement by using QICPIC equipment both 0.2 bar and 1.0 bar vacuumprocessing, and the size distribution curves were obtained forcomparison. FIG. 3 shows curve 1 corresponding to size distributionmeasured at 0.2 bar for a hemostatic aggregates specimen made with ahumidification step applied to ORC intermediate fine fiber prior toroller compaction. Curve 1 a shows size distribution measured at 1.0 barfor the same hemostatic aggregates specimen. The data indicates thatelevated processing pressure at 1.0 bar vacuum results in substantiallythe same size distribution or in minimal changes to size distributionfor hemostatic aggregates specimen made with a humidification stepapplied to ORC intermediate fine fiber prior to roller compaction. d50has varied from 190 to 199 microns only.

FIG. 4 shows the same testing performed for specimens manufacturedwithout humidification step. FIG. 4 shows curve 1 corresponding to sizedistribution measured at 0.2 bar for an hemostatic aggregates specimenmade without humidification step applied to ORC intermediate fine fiberprior to roller compaction. Curve 1 a shows size distribution measuredat 1.0 bar for the same hemostatic aggregates specimen. The dataindicates that elevated processing pressure at 1.0 bar vacuum results insubstantial change in size distribution for hemostatic aggregatesspecimen made no humidification step applied to ORC intermediate finefiber prior to roller compaction. d50 has substantially changed from 147to 84 microns indicating that the size of hemostatic aggregates shown inFIG. 4 is severely diminished when the pressure is increased.

High pressure treatment challenge can be related to hemostaticaggregates delivery via various delivery devices, including gas-assisteddelivery. Advantageously, according to one aspect of the presentinvention, hemostatic aggregates have substantially no change in sizedistribution when subject to processing at 1.0 bar vacuum. Importantly,excessive mechanical agitation or collisional forces can detrimentallyaffect the hemostatic aggregates size distribution and thus affect thehemostatic efficacy. The collisional forces generated in a SympatecQICPIC experiment is indicative of the sensitivity of hemostaticaggregates to pressure and can be used to qualitatively determinerelative stabilities.

Example 4. Hemostatic Properties

In another aspect of the present invention, the hemostatic aggregatesare shown to have superior hemostatic or blood clotting properties whentested in-vitro. Using the manufacturing techniques explained above,hemostatic aggregates samples were prepared with and withouthumidification step, with all other processing steps being equal. Somespecimens were the also subjected to the vibratory challenge asdescribed above.

Fresh porcine blood was placed in several 4.5 mL test tubes (BDVacutainer) with a 3.2% buffered sodium citrate solution and dilutedwith saline solution (0.9% NaCl USP) with a ratio of 2.5/1 (v/v). 1 mLof this blood solution was then placed into a 7 mL glass vial followedby the application of 100 mg of each hemostatic aggregates sample andlet standing for 2 minutes prior to evaluation. The vial was thenflipped up-side-down allowing any non-clotted blood to exit the vialinto a collecting receptacle. The remaining residues and clotted bloodin each vial were then evaluated by weight. Each sample was tested intriplicate. The results are shown in Table 2.

TABLE 2 % remaining in vial Test Test Test OBSER- Specimen I II III AVG.VATIONS Control: No hemostatic 5.43 4.38 2.70 4.17 No aggregatesadded(blood only) clotting hemostatic aggregates 98.21 95.77 97.38 97.12Excellent specimen made with a clotting humidification step applied toORC intermediate fine fiber prior to roller compaction hemostaticaggregates 97.05 91.97 93.64 94.22 Excellent specimen made with aclotting humidification step After applied to ORC intermediate vibrationfine fiber prior to roller compaction, then subjected to vibratorychallenge hemostatic aggregates 98.82 97.14 94.58 96.85 Excellentspecimen made with no clotting humidification step hemostatic aggregates14.30 15.38 12.88 14.19 Poor specimen made with no clottinghumidification step, After then subjected to vibratory vibrationchallenge

Analysis of data indicates that hemostatic aggregates specimens madewith a humidification step applied to ORC intermediate fine fiber priorto roller compaction exhibited excellent in vitro blood clotting, evenafter subjected to the vibratory challenge. On the contrary, whilehemostatic aggregates specimen made with no humidification stepexhibited excellent in vitro blood clotting, the same specimen afterbeing subjected to the vibratory challenge exhibited poor in vitroclotting. According to one aspect of the present invention, mechanicalstability of hemostatic aggregates results in sustained hemostaticproperties.

Example 5

Referring to Table 3, showing parameters of hemostatic aggregatesobtained in different batches, with parameters reported as averages ofthree tests. Process parameters were similar, with different feedingmaterial (intermediate fine fiber). Blood clotting was measured usingmethods described above. Bulk density and size distributions ofhemostatic aggregates were measured using methods described above.

As can be seen from Table 3, good clotting is achieved for hemostaticaggregates having a value of sphericity (sh50) equal of higher thanabout 0.6. The best clotting, i.e. with over 80% of blood remaining invial, was achieved for hemostatic aggregates having a value for itssphericity (sh50) of at least about 0.7 and a bulk density above 0.5(g/ml). It is noted that smaller sizes of feeding material resulted inhemostatic aggregates having these properties. Analysis of dataindicates that hemostatic aggregates specimens made with ahumidification step applied to ORC intermediate fine fiber prior toroller compaction and having bulk density above 0.5 exhibited excellentin vitro blood clotting. Analysis of data further indicates thathemostatic aggregates specimens made with a humidification step appliedto ORC intermediate fine fiber prior to roller compaction and havingsphericity (sh50) above 0.7 exhibited excellent in vitro blood clotting.

Based on the data in Table 3, hemostatic aggregates of the presentinvention have average sphericity above 0.6, preferably above 0.65, morepreferably above 0.7, most preferably above 0.75.

TABLE 3 Avg. (Int. Fine Avg % Bulk Fiber) (blood Avg. Density d(15)d(50) d(90) [d50, d90, Batch remaining) Sphericity (g/ml) (microns)(microns) (microns) microns] Control 5.51 N/A A 30.64 0.56 0.321 118 221372 [128, 271] B 59.81 0.59 0.331 113 203 357 [133, 268] C 63.16 0.610.365 132 228 368 D 71.61 0.67 0.423 85 178 328  [96, 200] E 85.45 0.730.510 121 206 340 F 96.80 0.76 0.525 111 178 307 G 96.71 0.79 0.528 145209 320  [65, 122]

Note: for line G material, the intermediate fine fiber was made usingthe ball mill process. Ball milling method to convert fabric tointermediate ORC fine fibers is described as the follows. 50 g ofpre-cut ORC fabric (2″×2″) was ball milled with 12 high-density Zirconiaballs 20 mm in diameter (Glen Mills Inc., Clifton, N.J., USA) by placingthe balls and the samples in a 500 mL grinding jar. The jar was clampedinto latching brackets and then counterbalanced on a planetary ball millPM100 (Retsch, Inc., Newtown, Pa., USA). The milling was then performedbi-directionally at 450 rpm for 20 minutes.

A linear regression and plot can be generated for d(50)[y=−301.03x+301.92, where R² is 0.950] and d(90) [y=−680.11x−659.02,where R² is 0.9887] for the source intermediate fine fiber relative tothe resulting hemostatic aggregates sphericity. Finer intermediate finefiber results in higher sphericity of hemostatic aggregates, such aswith intermediate fine powder having d(50) about 65 microns and d(90)about 120 microns, the sphericity of hemostatic aggregates is about 0.8.The same correlations are seen in Table 3.

As shown in Table 3, for d(50) and d(90) of intermediate fine powderbeing 96 and higher (i.e. 96 to about 130 for d(50) and 200 to about 270for d(90), the resulting blood clotting was 70%-30% and the sphericitywas 0.56-0.67. For d(50) and d(90) of intermediate fine powder beinglower than 96 (i.e. 35 for d(50)) and lower than 200 (i.e. 122 ford(90)), the resulting blood clotting was above 80% and the sphericitywas above 0.7. Smooth edged hemostatic aggregates, especially thosehaving a sphericity that approaches 1, flow well in applicator orsprayer, while spikey hemostatic aggregates flowed less-well inapplicator.

Referring now to FIG. 5, the results of hemostatic testing of thehemostatic aggregates shown in comparison with other hemostaticmaterials. A swine punch biopsy liver defect model was used. The testmaterials were hemostatic aggregates (referenced as bar A on the chart);plant based absorbable microporous polysaccharide hemostatic powderderived from purified plant starch (referenced as bar B); and plantstarch powder forming hydrophilic, adhesive hemostatic polymersconsisting of absorbable polysaccharides (referenced as bar C on thechart).

Test Method: 6-mm diameter by 3-mm deep defects were created using abiopsy punch. The site was allowed to bleed for several seconds prior toproduct application. The defect trial site was scored as hemostatic(Pass) if hemostasis was achieved in ≦10 minutes and maintained for 1minute without occlusive pressure, non-hemostatic sites were scored“Fail”. Time to Hemostasis (TTH) was measured for Pass sites. As can beseen from the FIG. 5, hemostatic aggregates produced a significantlylower TTH than comparative materials, 89% faster TTH than material B,and 93% faster TTH than material C with a p-value <0.001 in both cases.Bar D on the chart corresponds to negative control, i.e. bleeding whereno hemostatic agent was applied.

Example 6

Particle size distributions were obtained for the ORC aggregates andfine fibers. A typical aggregate material had volume weighted FeretMinimum D(15), D(50), and D(90) values of 111, 178, and 307 microns.This powder also had a sphericity, Sh(50)=0.76. Typical ORC fine fibershad length weighted fiber length D(10), D(50), and D(90) values of 30,72, and 128 microns.

Particle sizes and shapes were obtained with a Sympatec QICPIC imageanalyzer (Sympatec GMBH, Clausthal-Zellerfield, Germany). It has acamera resolution of 1024×1024 pixels with a pixel size of 10×10 μm².Its measurement range is from 5 to 1705 μm. A VIBRI/L vibratory feederwas used to introduce solid particles into a RODOS/L disperser. Imagesof the dispersed particles were then obtained in the QICPIC with acamera frame rate of 450 fps. A Feret min Q3 method was used tocalculate the particle sizes of the aggregates while a Sympatec LEFI Q1algorithm was used to determine the fiber lengths of the fibers.

The sphericity [Sh(50)] of the median-diameter aggregates was determinedby the Sympatec QICPIC method using the ratio of the perimeter P of theequivalent circle (P_(EQPC)) to the real perimeter (P_(real)), in whichA=area of the particle, shown in the equationS=(P_(EQPC))/(P_(real))=2(πA)^(1/2)/(P_(real)). The area of theequivalent projection circle has the same area as the projection area ofthe real particle.

Surface Area and Surface Wettability

Further characterizations of the materials were performed measuringsurface area and wettability of each hemostatic material. Wettabilityprovides a relative measure of surface polarity, and therefore theextent of hydrophilic or hydrophobic behavior of a material with wholeblood. Surface area analyses were performed with inverse gaschromatography (Surface Measurement Systems Model IGC-SEA, Alperton,UK). Approximately 750 mg of each sample was packed into individualsilanized glass columns (300 mm long by 4-mm inner diameter). Eachcolumn was conditioned with helium gas for 60 minutes at 37° C. and 0%relative humidity. All experiments were conducted at 37° C., with 10mL/min total flow rate of helium, using methane for dead volumecorrections. The Brunauer, Emmett, and Teller (BET) model was used forsurface area determinations, based on sorption isotherms with HPLC-gradedecane (Sigma-Aldrich, St Louis, Mo., USA) using the chromatograph inpulse sorption method.

The Brunauer, Emmett, and Teller (BET) surface areas for ORC aggregates[Sh(50)=0.76], ORC aggregates [Sh(50)=0.51], ORC fine fibers, and starchbased spheres are shown in Table 4. ORC aggregates with sphericityvalues of 0.51 and 0.76 had surface areas of 0.67 m²/g and 0.40 m²/g,respectively. ORC aggregates and fine fibers belong to the same familyof oxidized regenerated cellulose but ORC aggregates had a lower surfacearea/mass ratio. It was also found that ORC aggregates with lowersphericity values had higher surface areas than aggregates with highersphericity values if they had similar particle size distributions. Incontrast to the ORC powders, the starch-based spheres had the highestsurface area of the four materials.

TABLE 4 Sphericity and Surface Area of Test Materials Test materialSphericity Sh(50) Surface Area m²/g ORC aggregates 0.76 0.40 ORCaggregates 0.51 0.67 ORC fine fibers N.A. 1.17 Starch-based spheres 0.932.03

Analysis of Table 4 indicates that ORC aggregates with high sphericityvalues had much lower surface area vs. ORC fine fibers and ORCaggregates of low sphericity. ORC aggregates with high sphericity hadsurface area 1.5 times lower vs. ORC aggregates of low sphericity andclose to 3 times lower vs. ORC fine fiber.

The wettability or hydrophilicity of the test materials was determinedby dividing the acid-base surface energy by the total surface energy(γ^(AB)/γ^(T)). The surface energy profile was determined by mappingtechniques in which the specific free energies of desorption weredetermined by polarization. The dispersive surface energy component(γ^(D)) was measured by the method of Dorris and Gray using nonpolarHPLC grade probes: decane, nonane, octane, and heptane (Sigma-Aldrich,St Louis, Mo., USA). The acid-base surface energy component (γ_(s)^(AB)) was determined using the Good-van Oss-Chaudhury (GvOC) model, inwhich the acid-base component is taken as the geometric mean of theLewis acid parameter (γ_(s) ⁻) and Lewis base parameter (y_(s) ⁺). Thetotal surface energy (y^(T)) is the sum of the dispersive surface energyand the acid-base surface energy (y^(T)=γ^(D)+γ_(s) ^(AB)). Becauseγ_(blood) ^(AB) values were not available, the above equations weresimplified to calculate the works of adhesion and cohesion from thetotal surface energy values only, using the surface tension value forblood (γ_(blood) ^(T)) at 37° C.=52.6 mJ/m². The surface wettabilityresults are presented in Table 5.

TABLE 5 Surface wettability Surface Wettability ORC Aggregates Sh(50) =0.76 0.0384 ORC Aggregates Sh(50) = 0.51 0.0746 ORC Fine Fibers 0.110Starch-based Spheres 0.130

Analysis of Table 5 indicates that ORC aggregates with high sphericityvalues had much lower wettability vs. ORC fine fibers and ORC aggregatesof low sphericity. ORC aggregates with high sphericity had wettabilityalmost 2 times lower vs. ORC aggregates of low sphericity and close to 3times lower vs. ORC fine fiber.

Density

The “true density” of the materials was obtained by the gas pycnometer.The results are presented in Table 6. While densities of ORC materialsand starch spheres tested are all higher than water density of 1.0g/cm3, it is observed that interactions with blood were different. Onlythe aggregates of high sphericity have immediately penetrated the bloodsurface and initiated rapid clotting. Lower sphericity aggregates aswell as fine ORC fibers predominantly or partially stayed on the surfaceof blood as will be discussed below. In fact the true densities of alltested ORC aggregates and fine fibers are close, but high sphericity ORCaggregates exhibited immediate penetration of the blood surface.

TABLE 6 True Density of tested materials Material Density (g/cm3)Starch-based Spheres sample 1 1.3009 Starch-based Spheres sample 21.2987 Starch-based Spheres sample 3 1.299 Low sphericity ORC aggregatesample 1 1.5457 Low sphericity ORC aggregate sample 2 1.5451 Lowsphericity ORC aggregate sample 3 1.5451 ORC Fine Fiber sample 1 1.5313ORC Fine Fiber sample 2 1.5316 ORC Fine Fiber sample 3 1.5313 Highsphericity (0.76) ORC aggregate sample 1 1.5874 High sphericity (0.76)ORC aggregate sample 2 1.5875 High sphericity (0.76) ORC aggregatesample 3 1.5873

Despite similar density, ORC materials exhibited surprisingly differentpatterns of interactions with blood. The ORC fine fibers primarilyfloated on the surface of the blood with little penetration. The ORC lowsphericity aggregates exhibited some penetration but not as deep as thehigh sphericity aggregates.

The ability to penetrate into the blood appears to be directly relatedto the surface areas of the ORC materials. Higher surface area resultedin less penetration. Lower surface area materials will sink more rapidlyinto the blood. Wettability is another distinguishing feature of thesethree materials. The ORC fine fibers and low sphericity aggregates haveslightly higher wettability values than that of high sphericityaggregates. They are more hydrophilic. Powders with high surface areasand wettability values will interact with blood more rapidly than thosewith low surface areas and wettability values. Since the rate ofgelation of ORC and blood is relatively fast, powders with highersurface areas and wettability are not able to penetrate into the bloodand will remain near the surface. On the other hand, lower surface areapowders with low wettability will be able to interact with a largervolume of blood, resulting in better clots.

The starch based spheres have the highest surface area of all thematerials and their degree of penetration into blood was minimal.

Example 7. In Vitro Clotting. Further Hemostatic Evaluations

Fresh porcine blood was collected in 4.5-mL Vacutainer tubes (Becton,Dickinson and Company, Franklin Lakes, N.J., USA), with a 3.2% bufferedsodium citrate solution. A 1-mL aliquot of diluted blood was thentransferred to a 7-mL vial, after which 100 mg of each test article wasapplied. Clotting was allowed to proceed for 2 minutes at roomtemperature. The vial was capped, flipped upside down and placed on atapped density analyzer (Quantachrome Autotap EC148; QuantachromeInstruments, Boynton Beach, Fla., USA) and tapped mechanically 5 times.After 2 minutes the cap was removed, unclotted material drained bygravity, and the remaining residue in each vial was calculated byweight. Six replicates were performed for each sample.

The hemostatic activity of the ORC aggregates prepared at 2 sphericityvalues [Sh(50)=0.51 and Sh(50)=0.76], the ORC fine fibers from which theaggregates were derived, and a commercially available hemostat composedof starch-based spheres were examined. This investigation was initiatedto determine how overall sphericity of the ORC test materials affectedclotting and how these experimental products compared with an approvedabsorbable hemostat.

Samples were evaluated prior to and through up to 2 minutes afteraddition of 100 mg of each hemostat. In each panel, tube #1 was anuntreated control, tube #2 was treated with starch-based spheres, tube#3 was treated with ORC fine fibers, tube #4 was treated with lowsphericity ORC aggregates [Sh(50)=0.51], and tube #5 was treated withhigh sphericity ORC aggregates [Sh(50)=0.76].

It was observed that within seconds there were visible differences inthe activity of the test materials. The ORC aggregates with highsphericity [Sh(50)=0.76] immediately penetrated the surface of the bloodand initiated coagulation. The ORC aggregates with less sphericity[Sh(50)=0.51] penetrated, but to a lesser extent; and the ORC finefibers (essentially aspherical) remained somewhat superficial on thesurface of the liquid blood. The starch-based spheres remained on top ofthe blood surface and did not penetrate into the liquid. This indicatedthat a high degree of sphericity contributed to the blood-penetratingproperties of ORC aggregates. However, sphericity itself was not theonly factor affecting penetration, as the starch-based spheres were theleast penetrating and most spherical material tested [Sh(50)=0.93].

At 2 minutes there were visible differences in the clotting activity ofthe test materials. All the blood in the vial treated with highsphericity ORC aggregates was fully clotted, evidenced by the darkreddish-black color that is characteristic of ORC clots. The bloodtreated with low sphericity ORC aggregates and ORC fine fibers appearedless involved, and the blood treated with starch-based spheres appearedabout the same as untreated control blood. When the vials were inverted,only the high sphericity ORC aggregates appeared to produce a robust,adherent clot. There was no coagulation in the control tube containinguntreated blood. The high sphericity ORC aggregates produced a fullyinvolved clot that adhered to the vial. The low sphericity ORCaggregates produced a less adherent clot, and the ORC fine fibersproduced a modest clot. There was almost no clot in the tube treatedwith starch-based spheres.

The clotting efficacy was quantified by comparing the mass of the bloodin the vials before and after inversion. Vials were inverted,mechanically tapped 5 times with a tapped density analyzer, and allowedto rest for 2 minutes; unclotted blood simply dripped out from thebottom of the vial, and the remaining residue in each vial wascalculated by weight; each sample was tested in 6 replicates. Theresults of this testing are shown in FIG. 6. The clotting efficacy forthe high and low sphericity ORC aggregates was 95% and 38%,respectively. The clotting efficacy for ORC fine fibers and starch-basedspheres was 26% and 19%, respectively. Untreated blood only retained 4%of its weight as a clot. Error bars are ±standard deviation. Highsphericity ORC aggregates had the greatest clotting efficacy.

Example 8. In Vitro Clotting. Effect of Aggregate Sphericity onCoagulation Efficacy

Aggregates with several different sphericity values were produced andcompared. Comparing aggregates with similar particle size distribution,more spherical aggregates had a smaller surface area and had the highestclotting efficacy. In vitro coagulation assays were performed on batchesof ORC aggregates with sphericity values ranging from 0.51 to 0.79. Theresults are presented in FIG. 7, indicating that more sphericalaggregates had greater clotting efficacy than less spherical forms. At asphericity of 0.79, clotting efficacy was nearly 96%, whereas at asphericity of 0.51 the efficacy was less than 33%. Error bars are±standard deviation. Sphericity over 0.65, more preferably over 0.70,most preferably over 0.75 is preferred for high efficacy clotting.

Example 9. In Vivo Hemostasis

A long-term stability study that evaluated the effects of storageconditions and accelerated aging on hemostatic performance in a liverpunch biopsy model in swine was conducted. Within the larger study, itwas possible to compare the effect of ORC aggregate sphericity[Sh(50)=0.56 or Sh(50)=0.76] on hemostatic efficacy.

This study used five female Yorkshire Cross pigs weighing 54 to 57 kg.Biopsy punch defects were created using a 6-mm biopsy punch devicemarked to a depth stop of approximately 3 mm with surgical tape. Abiopsy punch was used to incise the parenchymal surface of the liver atan angle perpendicular to the tissue using a gentle twisting motion.Once the tissue was incised to the required 3-mm depth, the punch wasremoved. The tissue in the center of the punch site was removed usingforceps and surgical scissors and the assigned treatment was applied.

After a trial biopsy punch site was created, it was blotted with gauzeand the appropriate test article was applied to the site. A drynonadherent wound dressing (e.g., Telfa™ Non-Adherent Dressing) wasapplied on top of the test material followed by digital pressureensuring that adequate and even tamponade was applied to the site.

Pressure was initially held for 30 seconds followed by removal of thenonadherent dressing and a 30-second evaluation for hemostasis. Whenbleeding occurred during the initial evaluation period, pressure wasimmediately reapplied using a nonadherent wound dressing for anadditional 30 seconds followed by another 30-second evaluation forhemostasis up to a total time of 2 minutes after product application.When bleeding did not occur within the 30-second observation period, thetime to hemostasis was noted as the time when the last applied tamponadewas released. Any site that achieved hemostasis within 2 minutes wasthen lavaged with up to 10 mL of saline and observed for durable(maintained) hemostasis over another 30-second observation period. Ifbleeding occurred following lavage, durable hemostasis was noted as“fail,” and the surgeon used remedial measures to control bleedingbefore continuing with the testing period. If hemostasis was maintainedduring the 30-second observation period following lavage, durablehemostasis was noted as “pass.” If during the testing period tamponadeand observation periods continued longer than 2 minutes, i.e.,hemostasis was not achieved, the site was aborted and time to hemostasiswas recorded as more than 2 minutes in the raw data. This occurred onlyat negative control sites. This procedure was repeated with each testarticle as indicated. There was no attempt to reapply an article ifthere was a failure to achieve hemostasis when the article was appliedsuccessfully. Negative control sites were untreated.

The differences in in vivo hemostatic efficacy with regard to sphericitywere observable that paralleled the in vitro coagulation results. Allsites treated with ORC aggregates [Sh(50)=0.56, n=16; Sh(50)=0.76, n=12]had a median time to hemostasis of 30 seconds, and 100% of sites werefully hemostatic within 2 minutes. However, 38% of sites treated withlow sphericity ORC aggregates had delayed bleeding, necessitatingremedial application of another ORC material (ORC snow) to controlsignificant hemorrhage that occurred after the sample had been testedand classified as successfully hemostatic.

These observations confirmed in vitro data indicating that theaggregates with greater sphericity were more effective hemostaticagents.

In further aspects of the present invention, the hemostatic aggregatescan be combined with various additives to further improve the hemostaticproperties, wound healing properties, and handling properties, utilizingadditives known to these skilled in the art, including: hemostaticadditives, such as gelatin, collagen, cellulose, chitosan,polysaccharides, starch, CMC, calcium salts; biologics based hemostaticagents as exemplified by thrombin, fibrinogen, and fibrin, additionalbiologics hemostatic agents include, without limitation, procoagulantenzymes, proteins and peptides, each such agent can be naturallyoccurring, recombinant, or synthetic, and may be further selected fromthe group consisting of fibronectin, heparinase, Factor X/Xa, FactorVII/VIIa, Factor IX/IXa, Factor XI/XIa, Factor XII/XIIa, tissue factor,batroxobin, ancrod, ecarin, von Willebrand Factor, albumin, plateletsurface glycoproteins, vasopressin and vasopressin analogs, epinephrine,selectin, procoagulant venom, plasminogen activator inhibitor, plateletactivating agents, synthetic peptides having hemostatic activity,derivatives of the above and any combination thereof. Preferred biologichemostatic agents that can used in combination with the ball-milled ORCparticles are thrombin, fibrinogen and fibrin; Anti-infective agents,such as chlorhexidine gluconate (CHG), triclosan, silver, and similaranti-bacterial/microbial agents that are known in the art; and additivesthat increase the stickiness of the hemostat; diluents, salinesolutions, and similar additives that known in the art.

Having shown and described various versions in the present disclosure,further adaptations of the methods and systems described herein may beaccomplished by appropriate modifications by one of ordinary skill inthe art without departing from the scope of the present invention.Several of such potential modifications have been mentioned, and otherswill be apparent to those skilled in the art. For instance, theexamples, versions, geometrics, materials, dimensions, ratios, steps,and the like discussed above are illustrative and are not required.Accordingly, the scope of the present invention should be considered interms of the following claims and is understood not to be limited to thedetails of structure and operation shown and described in thespecification and drawings.

I/We claim:
 1. A method of making a plurality of hemostatic aggregates comprising the steps of: a) milling a cellulosic source material to form an intermediate fine fibers; b) humidifying the intermediate fine fibers; c) roller compacting the intermediate fine fibers to form hemostatic aggregates; d) sieving the hemostatic aggregates; e) dehumidifying the hemostatic aggregates; and f) optionally dosing the resulting hemostatic aggregates into storage containers or into delivery devices
 2. The method of claim 1, wherein, the step a) is preceded by a step of slitting and cutting the cellulosic source material forming pieces acceptable for milling in step a).
 3. The method of claim 2, wherein, the step a) is a two-part process with the second part is performed in an air classifier or ball mill process.
 4. The method of claim 3, wherein the second part is repeated three times.
 5. The method of claim 4, wherein the intermediate fine fibers have a size distribution of d50 less than about 100 microns and d90 of less than about 180 microns.
 6. The method of claim 1, wherein, in step b) the intermediate fine fibers are humidified to water content of between 11.0% and 20% by weight.
 7. The method of claim 1, wherein said step c) is performed by compacting the intermediate fine fibers into a compacted material which is then subjected to pre-breaking, followed by a step of final milling.
 8. The method of claim 7, wherein said compacting of the intermediate fine fibers is performed at a roller pressure of at least 125 bars.
 9. The method of claim 7, wherein said compacting the intermediate fine fibers is performed at a roller force of at least 26.0 kN/cm
 10. The method of claim 1, wherein said step d) is performed to select a targeted hemostatic aggregates fraction having dimensions of 75-300 μm by screen sieving.
 11. The method of claim 1, wherein said step d) is performed to select a targeted hemostatic aggregates fraction characterized by a size distribution such that d15 greater than or equal to ≧80 microns, d50 is from 140 to 250 microns and d90 less than or equal to ≦370 microns.
 12. The method of claim 1, wherein said step e) is performed to produce hemostatic aggregates having a moisture content of less than 5.5% determined by loss on drying.
 13. The method of claim 12, wherein said step e) is performed to hemostatic aggregates having moisture content of less than 2% determined by loss on drying.
 14. The method of claim 1 wherein the source material is oxidized regenerated cellulosic fabric, oxidized regenerated cellulose non-woven fabric, shredded oxidized regenerated cellulosic material or combinations thereof.
 15. The method of claim 1 wherein the source material further comprises an additive selected from the group consisting of carboxymethyl cellulose, calcium salt, an anti-infective agent, a hemostasis promoting agent, gelatin, collagen, or combinations thereof.
 16. The method of claim 1 further comprising a step of admixing an additive prior to step a), or prior to step b) by admixing the additive to the intermediate fine powder; or prior to step c) by admixing the additive to the humidified intermediate fine powder; or prior to step e) by admixing the additive to hemostatic aggregates prior to drying or prior to step f) by admixing the additive to hemostatic aggregates prior to dosing.
 17. A method of treating a wound comprising applying the hemostatic aggregates resulting from claim 1 onto and/or into the wound of a patient.
 18. Hemostatic particulate aggregates comprising a plurality of interconnected individual cellulosic fibrils having in aggregate form a sphericity of at least 0.5, a dimension along its longest axis that is less than about 500 microns and greater than about 50 microns.
 19. Hemostatic particulate aggregates comprising a plurality of interconnected individual cellulosic fibrils having in aggregate form a sphericity of at least 0.6, a dimension along its longest axis that is less than about 500 microns and greater than about 50 microns.
 20. The hemostatic aggregates of claim 18, wherein said hemostatic aggregates have a size distribution profile with d15 greater than about 80 microns, d50 from about 140 to 250 microns, d90 less than about 370 microns, a bulk density greater than 0.45 g/mL, and sphericity (sh50) equal or greater than 0.7.
 21. The hemostatic aggregates of claim 18 having substantially no size distribution changes or minimal size distribution changes after subjected to a vibratory challenge.
 22. The hemostatic aggregate of claim 21 wherein the size distribution profile of the hemostatic aggregates as measured by d50 does not fall below 100 microns.
 23. The hemostatic aggregates of claim 22, wherein said size distribution changes are characterized by a QICPIC optical sensor at 0.2 bars.
 24. The hemostatic aggregates of claim 22, wherein said mechanical stability is characterized by the hemostatic aggregates having substantially no size distribution changes or minimal size distribution changes after subjected to processing at 1.0 bar vacuum.
 25. A hemostatic aggregate that has been milled, humidified, roller compacted, and dried cellulosic material.
 26. A method of treating a wound comprising the step of applying the hemostatic aggregates of claim 18 onto and/or into the wound of a patient. 